ABSTRACT Diabetic retinopathy remains a major cause of blindness, and despite cutting edge research in the field, the molecular mechanism of its pathogenesis remains unclear. Our studies have documented a critical role of matrix metalloproteinase 9 (MMP-9) in diabetic retinopathy, and have demonstrated that cytosolic MMP-9 activation is an early event, that is followed by its mitochondrial accumulation, mitochondrial dysfunction and mtDNA damage, initiating a vicious cycle of free radicals. Epigenetic modifications play a critical role in MMP-9 transcription, and in diabetes, MMP-9 promoter DNA undergoes dynamic methylation-hydroxymenthlation and histone modifications. MMP-9 is also regulated by homocysteine, a thiol-containing non-protein amino acid, and diabetic patients have elevated plasma homocysteine levels. Increased homocysteine is implicated in cellular and metabolic abnormalities including mitochondrial damage and epigenetic modifications. Homocysteine is also a precursor of hydrogen sulfide (H2S), and due to impaired homocysteine metabolism, plasma levels of H2S are decreased in diabetic patients. Based on these, our central hypothesis is that in diabetes, high homocysteine activates MMP-9 and disturbs mitochondrial dynamics, and the damaged mitochondria accelerates apoptosis resulting in the development of diabetic retinopathy. Aim 1 will investigate the mechanism(s) by which homocysteine activates MMP-9 in diabetes, and the model predicts that high homocysteine activates MMP-9 by (i) damaging interactions between MMP-9 and its tissue inhibitor, Timp1, and (ii) inducing epigenetic modifications and increasing the ratio of MMP-9-Timp1. Aim 2 will determine the mechanism(s) by which homocysteine impairs mitochondrial dynamics, and will test the hypothesis that homocysteine increases mitochondrial fragmentation, and dysfunctional mitophagy in diabetes fails to properly remove the fragmented mitochondria. Aim 3 will examine the therapeutic potential of regulating homocysteine-H2S metabolic balance on inhibition of diabetic retinopathy. The plan will employ in vitro (retinal endothelial cells) and in vivo (retinal microvessels from rodents) models of diabetic retinopathy, and will utilize fully optimized molecular biological and pharmacological approaches. Our overall goal is to identify novel regulatory mechanisms involved in the pathogenesis of diabetic retinopathy, specifically at the level of regulation of homocysteine-H2S. The proposal is based on a testable central hypothesis, and our proposed studies are innovative and carry a significant translational impact as they are expected to identify novel therapeutic targets to prevent the development and progression of diabetic retinopathy. This will offer patients additional therapeutic means to prevent/halt this sight-threatening complication of diabetes.